Alveoli are the gas-exchange units of lung. The process of alveolar development,
alveologenesis, is regulated by a complex network of signaling pathways that act on various cell types
including alveolar type I and II epithelial cells, fibroblasts and the vascular endothelium. Dysregulated
alveologenesis results in bronchopulmonary dysplasia in neonates and in adults, disrupted alveolar
regeneration is associated with chronic lung diseases including COPD and pulmonary fibrosis.
Therefore, visualizing alveologenesis is critical to understand lung homeostasis and for the
development of effective therapies for incurable lung diseases. We have developed a technique to
visualize alveologenesis in real-time using a combination of widefield microscopy and image
deconvolution of precision-cut lung slices. Here, we describe this live imaging technique in step-by-step
detail. This time-lapse imaging technique can be used to capture the dynamics of individual cells within
tissue slices over a long time period (up to 16 h), with minimal loss of fluorescence or cell toxicity.